Immunogold Labeling
Young barley leaves were cut transversely with a fresh razorblade into 1 mm diameter strips. The samples were then prepared for immunogold labelling (IGL) and transmission electron microscopy (TEM) according to Rubio et al. (2019). Briefly, they were fixed immediately in 4% paraformaldehyde + 0.5% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.0) overnight. After dehydration in ethanol the samples were infiltrated and embedded in LR White resin. The leaf samples were sectioned on a Leica UCT ultramicrotome and the sections (80 nm) collected on Ni grids coated with pyroxylin. After 1 h blocking in IGL buffer (Rubio et al. 2019) the grids were incubated for 2 h at room temperature in a polyclonal antibody (diluted 1:10 in IGL buffer) raised against barley WHIRLY1 in goat (Agrisera, Vannas, Sweden). After two washes in IGL buffer (10 min per wash) the grids were then incubated for 2 h in rabbit anti-goat IgG 10 nm gold (Aurion Immuno Gold Reagents & Accessories, Wageningen, Netherlands). The grids were finally washed twice in IGL buffer (5 min per wash) and 10 times in dH2O (30 s per wash). The immunogold labelled grids were viewed and photographed under a JEOL JEM1400 transmission electron microscope, and gold particles were counted on 8 - 10 representative images from each sample taken at the same magnification (x4000).